Ethyl pyruvate (EP), a simple ester of pyruvic acid, has been shown to exert robust neuroprotection in various neuropathological conditions, such as, cerebral ischemia and KA-induced seizure animal models. The neuroprotective effect of EP is attributable to the anti-inflammatory, anti-oxidative, and anti-apoptotic effects. In the present study, we investigated convergence of anti-inflammatory and anti-oxidative functions of EP and present a novel molecular mechanism underlying anti-inflammatory effects of EP, which is conveyed by p300, a transcriptional co-activator for both Nuclear factor E2-related factor 2 (Nrf2) and p65. In BV2 cells, a microglia cell line, EP induced translocation of Nrf2 from the cytosol to the nucleus and enhanced the expression of hemeoxygenase 1 (HO-1) in a dose-dependent manner and 1h incubation with 10mM EP increased HO-1 to 4.9-fold. Nrf2 was found to translocate from the cytosol to the nucleus beginning 30 min after EP-treatment and binds to the antioxidant response element (ARE) located on HO-1 promoter. Interestingly, LPS-induced inducible NO synthase (iNOS) induction was substantially suppressed in EP-pre-treated BV2 cells and it was reverted by Nrf2 knockdown. We found that EP-induced Nrf2 accumulation in the nucleus recruits p300, a transcriptional co-activator of both Nrf2 and p65, inhibiting p65-p300 interaction. Competition between Nrf2 and p65 for p300 binding was confirmed by glutathione S-transferase (GST) pull down assay and reporter gene analysis. These results demonstrate that EP induced nuclear translocation of Nrf2 which binds to ARE along with p300 and hampers iNOS expression via depleting p300 from p65. This is a novel anti-inflammatory mechanism conveyed by EP, which enhances protective effect by converging anti-inflammatory and anti-oxidative effects and might be applicable to various Nrf2-activating agents, such as phytochemicals.
Keywords: 12-O-tetradecanoylphorbol-13-acetate.; ARE; Anti-inflammation; CBP; CREB-binding protein; EP; Ethyl pyruvate; GCLM; GDNF; GSH; GST; HO-1; IL-1 β; LPS; NAD(P)H dehydrogenase, quinone 1; NF- κB; NQO-1; Nrf2; P300; P65; PMA; antioxidant response element; ethyl pyruvate; glial cell-derived neurotrophic factor; glutamate-cysteine ligase modifier; glutathione; glutathione S-transferase; hemeoxygenase 1; iNOS; inducible NO synthase; interleukin-1 beta; lipopolysaccharide; nuclear factor E2-related factor 2; nuclear factor kappa-light-chain-enhancer of activated B cells; p38 MAPK; p38 mitogen activated protein kinase.
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